Controlled cell death is critical for the life of a human; too much cell death can lead to diseases such a neurodegeneration and autoimmune deficiency syndrome (AIDS) while too little cell death can lead to cancer or autoimmune diseases. Recent studies have defined the pathway of cell death as "apoptosis" and have identified some of the biochemical steps involved.
Apoptosis is a homeostatic mechanism involved in the controlled death of obsolete cells during metamorphosis, differentiation, cell turnover, and hormone mediated deletion of thymocytes (Wyllie et al. Int. Rev. Cytol. 1980 68:251-306). Apoptosis has also been identified as the mechanism of cell killing during growth factor withdrawal (Rodriguez-Tarduchy et al. EMBO J. 1990 9:2997-3002; McConkey et al. J. Biol. Chem. 1990 265:3009-3011), T-cell deletion, treatment with many cytotoxic agents (Cohen, J. J. and Duke, R. C. J. Immunol. 1984 132:38-42; Barry et al. Biochem. Pharmacol. 1990 40:2353-2362; Kaufmann, S. H. Cancer Res. 1989 49:5870-5878; and McConkey et al. Science 1988 242:256-259), and following hypothermia (Barry et al. Biochem. Pharmacol. 1990 40:2353-2362; Lennon et al. Biochem. Soc. Trans. 1990 18:343-345; Takano et al. J. Pathol. 1991 163:329-336).
Central to the mechanism of apoptosis is internucleosomal DNA digestion by endogenous endonucleases. Mammalian cells contain a variety of endonucleases which could be involved in internucleosomal DNA digestion. However, it has been postulated that the primary endonuclease involved in apoptosis is a Ca.sup.2+ /Mg.sup.2+ -dependent endonuclease. Several Ca.sup.2+ /Mg.sup.2+ -dependent endonucleases have been identified, one of which is deoxyribonuclease I (DNase I), (Peitsch et al. EMBO J. 1993 12:371).
Recent experiments, however, indicate that DNase I may not be the primary endonuclease involved in apoptosis. It has been found that many cells do not contain this endonuclease. The role of DNase I, or any other Ca.sup.2+ /Mg.sup.2+ -dependent endonuclease is further unlikely, as often no increase, or only a minor increase, in Ca.sup.2+ levels occurs in apoptotic cells (Eastman, A. Cell Death and Differentiation 1994 1:7-9).
An alternate endonuclease that is active below pH 7.0 and has no apparent requirement for Ca.sup.2+ or Mg.sup.2+ has been detected (Barry, M. A. and Eastman, A. J. Natl Cancer Inst. 1990 82:749). This alternate endonuclease has been identified as deoxyribonuclease II (DNase II; Barry, M. A. and Eastman, A. Archives of Biochem and Biophys. 1993 300(1):440-450). It is believed that this enzyme is involved in the internucleosomal digestion or fragmentation of DNA which is one of the early steps in the pathway of apoptosis. Another report that has implicated DNase II in cell death involves lens fiber cell differentiation, a process where the cells lose their nuclei in a manner similar to apoptosis (Torriglia, A. et al. 1995 J. Biol. Chem. 270:28579-28585). In this process, the chromatin condenses and the cells degrade their genomic DNA. DNase II was found by immunocytochemistry to be localized in the cytoplasm but translocated to the nucleus of the fiber cell before degeneration. These findings implicate DNase II as the endonuclease responsible for genomic degradation observed during lens nuclear degeneration, and further support a role for this enzyme in mechanisms of cell death.
DNase II has now been isolated and purified and the amino acid sequence determined. Further, the DNA sequences for both the human and bovine proteins have now been cloned.